【摘要】 目的筛选一个新的锌指蛋白Mip1的DNA结合位点。方法将Mip1cDNA全长克隆入原核表达质粒pGEX-4T-1构建了Mip1的原核表达质粒pGEX-Mip1。用谷胱甘肽亲和层析纯化GST-Mip1融合蛋白。通过固定化的GST-Mip1,从寡核苷酸文库中俘获Mip1的结合的寡核苷酸片段;将所得寡核苷酸片段插入T-载体,通过测序、序列比对得到DNA结合位点。结果锌指蛋白Mip1的融合蛋白能在大肠杆菌中高效表达,能与固定化还原性谷胱甘肽很好地亲和而得到纯化;用固定化的Mip1融合蛋白俘获到了其结合的寡核苷酸序列,通过测序、序列比对,得到了Mip1的DNA结合位点。结论Mip1的DNA结合位点的核心序列为G/TCCTA,Mip1的DNA结合位点的成功确定为Mip1功能的深入研究打下了基础。更多还原

【Abstract】 Objective To screen the DNA binding site of a novel zinc finger protein named Mip1. Methods Mip1 full-length ORF was cloned into a bacterial expression vector pGEX-4T-1 to construct pGEX-Mip1. pGEX-Mip1 was overexpressed in E.coli M15 to generate soluble GST-Mip1 fusion protein,and the GST-Mip1 was purified by glutathione affinity chromatography and immobilized on Sepharose beads to capture putative target DNA binding site from the oligonucleotide library. The selected oligonucleotides were gel-purified and inserted into T vector for sequencing and the consensus sequence was derived by sequence alignment. Results GST-Mip1 fusion protein was over-expressed in E.coli M15 and purified by affinity chromatography. Using immobilized-Mip1 beads,a lot of oligonucleotides were fished out from the oligonucleotides library. A consensus sequence which may be the DNA binding site of Mip1,was derived from sequence alignment. Conclusion The DNA binding site of Mip1 may be the consensus sequence G/TCCTA.更多还原